patients. After a single dose of AZD9291 mesylate salt, followed by a 7 day washout and then 8 days of once daily 20 mg oral dosing in AURA Phase 1 study
(NCT01802632).
B and C. Serial computed tomography scans of the chest from patients before and after treatment with AZD9291 in a phase I trial. B, Images from a 57-year old Korean female patient diagnosed with Stage IV non-small cell lung cancer in May 2011. See main text for details. C, Images from a 57-year old British female never smoker diagnosed with Stage IV lung adenocarcinoma in December 2010. The patient was previously treated with first-line gefitinib for 14 months, achieving a partial response before eventually developing progressive disease. See main text for details.
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partial response before progressing immediately before study. Analysis of tumor tissue, from a biopsy taken immediately before AZD9291 study entry, using direct
dideoxynucleotide-based sequencing, revealed presence of a T790M mutation (data not shown). Tumor shrinkage on AZD9291 was 39.7% at scan 1, 48.3% by scan 2 (Figure 21B), remained at 48.3% at both scan 3 and scan 4, and was 51.7% at scan 5 (data not shown).
The second patient was a 57-year old white female never smoker from England diagnosed with Stage IV lung adenocarcinoma in December 2010. Analysis of tumor tissue obtained at diagnosis in a local molecular pathology lab using the Qiagen EGFR RGQ PCR test revealed evidence of exon 19 deletion and T790M mutations (data not shown). The patient was treated with first-line gefitinib, achieving a partial response before eventual progressive disease 14 months later, suggesting that the T790M mutation was present at only a low allele frequency. Re-biopsy prior to starting AZD9291 was not performed. At the cycle 1 day 15 assessments on AZD9291, the patient reported full resolution of pre-existing persistent nocturnal cough. Tumor
shrinkage was 38% at scan 1, 39.3% at scan 2, 56.7% by scan 3 (Figure 21C), 62% by scan 4, and 59.3% by scan 5 (data not shown). By Cycle 7 Day 1, the patient reported significant improvement in pre-existing hair and eyelash abnormalities which had developed during the immediately prior gefitinib therapy. Since this patient received AZD9291 after developing acquired resistance whilst on continuous gefitinib (after initial
> 6 months duration of partial response) with no intervening treatment, strict Jackman clinical criteria for acquired resistance were fulfilled (Jackman, Pao et al. 2010). No significant aberration of blood glucose levels were noted in either patient during the
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study. Both patients had duration of response of approximately 9 months and were progression-free on 20 mg/day AZD9291 for approximately 11 months, until disease progression by RECIST 1.1. Both patients continue to receive AZD9291 treatment on study as per protocol, as they continue to derive clinical benefit according to their treating physicians.
Discussion
Mutations in EGFR occur in 10-35% of NSCLCs and confer sensitivity to the EGFR TKIs erlotinib, gefitinib, and afatinib (Pao and Chmielecki 2010). In randomized studies, the median overall survival of patients with EGFR mutant lung cancer receiving first-line EGFR TKIs is ~19-36 months, while median progression free survival is about a year. In more than half of patients, tumor cells at the time of progression harbor a second-site T790M mutation, which confers resistance to these EGFR TKIs (Yun, Mengwasser et al. 2008). No specific treatments for patients with acquired resistance to current EGFR TKIs have yet been approved.
We describe here the identification, characterization and early clinical
development of AZD9291, a novel oral, irreversible, third generation TKI with a distinct profile from gefitinib, erlotinib, afatinib, and dacomitinib. Notably, AZD9291 has a distinct chemical structure from the other third-generation TKIs, WZ4002 (Zhou, Ercan et al.
2009) and CO-1686 (Walter, Sjin et al. 2013). Biochemical profiling together with in vitro cellular phosphorylation and phenotype studies have collectively shown that AZD9291 is highly potent against EGFR-mutant and T790M resistant EGFR mutants with a wide margin of selectivity against wild type EGFR activity and exhibits a high degree of selectivity against other kinases outside the EGFR family. Moreover, the profound anti-
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tumor activity of AZD9291 across xenograft and transgenic mutant T790M EGFR disease models in vivo suggests the potential to target T790M tumors following progression on early generation TKIs.
Prior to identification of “third generation” EGFR TKIs, the most promising targeted regimen in patients with acquired resistance had been the combination of afatinib plus cetuximab, which induced a 32% response rate and median progression- free survival of 4.7 months in a heavily pre-treated cohort (Janjigian et al) with a significant degree of skin and gastrointestinal (diarrhea) toxicity. In the phase I trial of AZD9291 in EGFR-mutant NSCLC patients that had progressed on earlier generation TKIs, evidence of efficacy has been seen at all doses studied so far, with AZD9291 induced partial radiographic responses in patients whose tumors were known to harbor T790M, from the first dose cohort onwards (Burtness, Anadkat et al. 2009). Rash and diarrhea were mostly mild and reported in only a minority of patients, consistent with low activity against wild-type EGFR. Out of the two confirmed partial responses described in this paper, in addition to both patients’ tumors harboring the T790M mutations
(according to local tests), one patient’s disease fits strict Jackman criteria for acquired resistance (Jackman, Pao et al. 2010), receiving the drug directly after prolonged response and progression on gefitinib. Thus, based on the above, AZD9291 has already demonstrated proof-of-principle clinical activity in patients with acquired
resistance for whom there are no approved targeted therapies. Similarly, results from a phase I trial with CO-1686 have also shown evidence of efficacy in TKI-resistant tumors harboring T790M, providing further proof of principle for potential use of “third
generation” TKIs in this setting. A surprising finding in the afatinib plus cetuximab study
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was that tumors with undetectable levels of T790M also displayed tumor shrinkage, suggesting that a T790M-independent but EGFR-dependent pathway of resistance exists. The current phase I study of AZD9291 will test whether the drug is effective in both T790M-positive and –negative cohorts, through planned dose expansion cohorts.
Full Phase I data will be presented at completion of study.
The existence of cell populations harboring T790M within a proportion of TKI- naive EGFR-mutant tumors has been reported, although the prevalence depends on the diagnostic assay being used. Studies using more conventional diagnostic assays have reported detection in about 2% of TKI-naive tumors (Sequist, Martins et al. 2008).
Recently, groups using more sensitive technologies have reported higher detection rates ranging from 40% to 60% (Costa, Molina et al. 2014), although it remains unclear whether all these observations are real or analytical artifacts (Ye, Zhu et al. 2013).
However, overall the data supports the hypothesis that T790M clones pre-exist in a proportion of EGFR-mutant tumors prior to TKI treatment. In addition to T790M, AZD9291 also potently inhibits sensitizing mutant EGFR across in vitro and in vivo disease models at similar potencies to T790M-mutant EGFR. Therefore, taken together, this supports the hypothesis that AZD9291 could also offer an attractive treatment option in EGFR-mutant TKI-naive patients, through targeting both sensitizing and T790M tumor cell populations that co-exist in a proportion of tumors, which may then lead to delayed disease progression and ultimately increased survival benefit. However, it remains to be determined what the optimal sequencing of EGFR TKIs will be and whether maximum benefit to most patients will be achieved through using AZD9291 after TKI progression or earlier in the treatment pathway.
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Patients harboring EGFR-mutant tumors often progress during TKI treatment due to growth of secondary brain metastases (Porta, Sanchez-Torres et al. 2011). Although there are reports of TKIs providing benefit in treatment of EGFR-mutant brain
metastases, current TKIs are believed to have generally poor properties for penetrating across the blood brain barrier (BBB), and thus their activity will be variable and
influenced by such factors as dose, level of BBB disruption and efflux transporter expression across individuals. Therefore, there is also a need for EGFR TKIs with improved brain penetrance. Quantitative whole body autoradiography (QWBA) studies in rat brain with [14C]AZD9291 have indicated AZD9291 had a brain-to-blood ratio of up to 2 over the first 24 hours, suggesting the potential of AZD9291 to penetrate the brain (data not shown). This is in contrast to [14C]gefitinib which had a maximum brain-to- blood ratio of only 0.2 (McKillop, Hutchison et al. 2004). Although further pre-clinical studies are required to explore the translatable potential of AZD9291 to target brain metastases, together with future clinical studies, the preliminary data look promising in this area.
Despite the potential of AZD9291 to prevent resistance via T790M, tumors are likely to engage alternative escape mechanisms. If TKIs such as AZD9291 become a prominent feature in the treatment of EGFR-mutant disease across multiple lines of therapy, it will be critical for future pre-clinical and clinical research to identify the most prevalent future resistance mechanisms. Consistent with its pharmacological profile, we have not observed acquired resistance to AZD9291 in vitro due to emergence of
T790M. It is also interesting to note that we have not yet seen resistance to AZD9291 due to direct mutation of cysteine 797 (data not shown), which would render the
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receptor refractory to irreversible agents, in an analogous manner T790M prevents inhibition by early generation drugs. Therefore, non-EGFR related resistance
mechanisms may become more dominant for agents such as AZD9291. Indeed, pre- clinical reports have suggested that “third generation” agents can induce switching to multiple signaling mechanisms that bypass EGFR dependency such as ERK and AKT pathways (Ercan, Xu et al. 2012; Cortot, Repellin et al. 2013; Walter, Sjin et al. 2013).
Since AZD9291 is structurally distinct, it will be critical to understand which resistance mechanisms are induced through treatment with AZD9291 and whether different “third generation” TKIs engage common escape mechanisms. Furthermore, identification of molecular mechanisms of resistance will support the investigation of strategies to combine additional novel targeted therapies with AZD9291 as a foundation EGFR TKI partner, to achieve potentially greater clinical benefit.
AZD9291 and its active circulating metabolite AZ5104 display similar and minimal off-target activity against other non-HER family kinases, but in vitro data
suggests the potential to target both HER2 and HER4 kinase activity. This property may be important as HER2 amplification may mediate acquired resistance to EGFR TKI in some cases (Takezawa, Pirazzoli et al. 2012). AZD9291 and AZ5104 also appear to be effective against other rare drug-sensitive EGFR mutants and potentially lung cancer- associated HER2 mutants, but like other EGFR TKIs are not potent against an EGFR exon 20 insertion. Further pre-clinical and clinical studies are required to understand the potential utility of AZD9291 in these additional molecular phenotypes.
Earlier generation EGFR TKIs have revolutionized the treatment of EGFR-mutant
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NSCLC and have demonstrated the power of precision medicine in genetically-defined tumors. However, toxicities related to wild-type EGFR and the emergence of resistance mechanisms have limited the effectiveness of these drugs. Third-generation EGFR TKI agents such as AZD9291 have the potential to overcome these limitations and improve markedly the treatment options to patients who have progressed on TKI treatment due to T790M. Evaluation of AZD9291 in the 1st line setting in patients with EGFR-mutant tumors should also be considered based on AZD9291’s third generation EGFR TKI profile. Moreover, if AZD9291 is confirmed to have a mild side effect profile together with its clinical efficacy and mechanistic hypothesis, this raises the option for
investigation as a foundation EGFR TKI for combinations with other therapies to provide further treatment options for patients.
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Merlin regulates EGFR expression in some, but not all, EGFR-mutant lung cancer cell lines
Introduction
Lung cancer is the leading cause of cancer-related deaths in the US and worldwide (Molina, Yang et al. 2008). Historically, lung cancers have been treated with cytotoxic chemotherapies, with mixed results. Over the past decade, tremendous advances in genomic technologies have enabled the identification of specific mutations that drive lung tumors. Such mutations are causally implicated in oncogenesis and positively selected for throughout tumor generation (Weinstein 2002; Stratton, Campbell et al. 2009). Importantly, some of these mutations can be targeted by drugs, matching the specific driver mutation of patient’s tumor to a specific drug. This personalized approach to cancer medicine often results in far better outcomes for patients, including both reduced tumor burden and decreased toxicity profile.
In 2004, mutations in the epidermal growth factor receptor (EGFR) were identified as an essential biomarker for sensitivity to first-generation anti-EGFR tyrosine kinase inhibitors (TKIs), erlotinib and gefitinib (Lynch, Bell et al. 2004) (Pao, Miller et al.
2004) (Paez, Janne et al. 2004). Patients whose tumors harbor activating mutations in EGFR now routinely receive erlotinib or gefitinib as first-line therapy; most patients will
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respond and experience decreased tumor burden. However, within a median of 9-16 months, all patients will invariably experience disease progression, defined as primary acquired resistance. Much work has focused on mechanisms of and treatment methods to overcome primary acquired resistance. One such therapy combines a second- generation EGFR TKI, afatinib, plus monoclonal anti-EGFR antibody, cetuximab (Regales, Gong et al. 2009). A phase Ib trial within patients with primary acquired resistance observed a 29% response rate to afatinib + cetuximab. Unfortunately, all responders will eventually fail second-line anti-EGFR targeted therapy and experience secondary acquired resistance.
We previously identified two of the first patients to experience secondary acquired resistance (Pirazzoli, Nebhan et al. 2014).In one patient, we identified two mutations in NF2 that were present at the point of disease progression, but not prior to starting afatinib+cetuximab. Mutations in merlin, the protein product of the NF2 gene, had not previously been reported in the context of acquired resistance. The first mutation, c.592C>T_p.R198* at frequency 0.15 of 631 reads, is a truncating mutation, while the second, c.811-2A>T: splice at 0.13 frequency of 1168 reads, is a splice site mutation expected to cause a deletion of eight amino acids. Both mutations are predicted to cause loss-of-function due to their location in the protein’s FERM domain, essential for merlin’s function and localization (Figures 3, 4). Mutations in NF2 piqued our interest as a potential mechanism of acquired resistance to afatinib+cetuximab because loss of merlin has been shown to activate EGFR signaling, at least in non- cancerous cells with wild-type EGFR (Curto, Cole et al. 2007). After we established that knockdown of merlin induces resistance to anti-EGFR agents in EGFR-mutant lung
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cancer cell lines, which may be abrogated by concurrent treatment with mTOR inhibitor (see Chapter III), we sought to understand the mechanism by which merlin regulates EGFR in EGFR-mutant cells.
Results
Merlin is expressed in EGFR-mutant lung cancer cell lines
Because the function of merlin is dependent on cell density, all experiments described herein were conducted at 100% total cell confluence at the time of cell lysis.
To begin evaluating the mechanism of merlin in EGFR-mutant lung cancer cells, we first evaluated a panel of EGFR-mutant cell lines for the presence of full-length merlin. As a control, we acquired the only commercially-available NF2-/- lung adenocarcinoma cell line, SW1573. This cell line does not harbor any EGFR mutation but does contain a KRAS G12C mutation. EGFR-mutant lung cancer cell lines evaluated included those harboring both exon 19 deletions and L858R mutations in EGFR. All cell lines harbored full-length merlin by immunoblot analysis; though protein levels were not totally consistent among cell lines, no patterns were identified (Figure 22, upper panels).
Because the NF2 mutations discovered in a patient were found in the context of acquired resistance to anti-EGFR agents, we next evaluated isogenic pairs of TKI- sensitive and -resistant EGFR-mutant cell lines for merlin loss. Resistant cell lines were developed in our lab by prolonged drug exposure of parental cells as previously described (Chmielecki, Foo et al. 2011). No evidence of merlin loss upon resistance to
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Figure 22. Merlin is expressed in EGFR-mutant lung cancer cell lines
Immunoblot ananalysis of endogenous merlin expression in EGFR-mutant TKI sensitive cell lines (top) and isogenic pairs (bottom) of TKI-sensitive (par, parental) and –resistant (/ER, erlotinib-resistant; /BR, afatinib-resistant; XLR, XL647-resistant). SW1573 is an wtEGFR, mtKRAS, NF2-/- lung adenocarcinoma cell line used as a control. Results are representative of at least three experiments per cell line.
actin merlin total EGFR
control lines exon 19 deletion lines L858R lines
actin total EGFR merlin
exon 19 deletion L858R NF2-/-
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drug was seen by immunoblot analysis within various isogenic pairs (Figure 22, lower panels).
Merlin expression is not affected by treatment with anti-EGFR targeted therapies
Next, we evaluated whether treatment with anti-EGFR agents affected levels of merlin in EGFR-mutant cell lines. PC9par and HCC827par cells were exposed to erlotinib (data not shown), afatinib or cetuximab over a 4-day time period (Figure 23). In all cases, drug was effective at reducing or eliminating phosphorylated EGFR. When treated with cetuximab, prolonged exposure resulted in the expected downregulation of total EGFR. However, in all cases, merlin levels remained stable, suggesting that merlin expression is not affected by short-term treatment with anti-EGFR tyrosine kinase inhibitors or an anti-EGFR antibody.
Merlin expression regulates EGFR expression in some EGFR-mutant cell lines
To explore further the mechanism of merlin regulation of EGFR in the context of EGFR-mutant cells, we transiently transfected an siNF2 construct into PC9par and HCC827par cell lines. In both cell lines, we observed efficient merlin knockdown.
However, we saw an unexpected decrease in total EGFR upon merlin knockdown in PC9par cells. This decrease in total EGFR was not observed in HCC827par cells, suggesting that the observation was not due to off-target effects against EGFR by the siNF2 construct. To further investigate, we conducted a time course analysis in which either a scrambled, siEGFR, or siNF2 construct was transfected into cells. We saw that knockdown of EGFR resulted in no change in merlin expression in both cell lines, suggesting that merlin expression is not dependent on EGFR expression. However, we
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Figure 23. Merlin expression is not decreased by treatment with anti-EGFR targeted therapies
Time course treatment of PC9par cells (top panels) and HCC827par cells (bottom panels) with 100nM afatinib (left panels) or 10µg/mL cetuximab (right panels). Results are representative of at least three experiments.
100nM afatinib
NT 5’ 15’ 30’ 1hr 3hr 6hr 12hr 24hr 48hr 72hr 96hr
merlin actin total EGFR pEGFR
(Y1068)
merlin actin total EGFR pEGFR
(Y1068)
NT 5’ 15’ 30’ 1hr 3hr 6hr 12hr 24hr 48hr 72hr 96hr
10µg/mL cetuximab
PC9parHCC827par
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Figure 24. Merlin expression regulates EGFR expression in some EGFR-mutant cell lines
Time course transfection of PC9par cells (top panel) or HCC827par cells (bottom panels) with scrambled siRNA construct, siEGFR, or siNF2 for 12-72hr timecourse of treatment. For all treatments, reverse transfection protocol was used to ensure cell confluence at the time of lysis. Results are representative of at least three experiments for each cell line/transfection condition.
actin total EGFR
merlin
12 24 48 72
scrambled siEGFRpool siNF2pool
12 24 48 72 12 24 48 72 No Tx
actin total EGFR
merlin
No Tx 12 24 48 72
scrambled siEGFRpool siNF2pool
12 24 48 72 12 24 48 72
PC9par
HCC827par